A natural mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody
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Abstract
Epitopes that are conserved among SARS-like coronaviruses are attractive targets for design of cross-reactive vaccines and therapeutics. CR3022 is a SARS-CoV neutralizing antibody to a highly conserved epitope on the receptor binding domain (RBD) on the spike protein that is able to cross-react with SARS-CoV-2, but with lower affinity. Using x-ray crystallography, mutagenesis, and binding experiments, we illustrate that of four amino acid differences in the CR3022 epitope between SARS-CoV-2 and SARS-CoV, a single mutation P384A fully determines the affinity difference. CR3022 does not neutralize SARS-CoV-2, but the increased affinity to SARS-CoV-2 P384A mutant now enables neutralization with a similar potency to SARS-CoV. We further investigated CR3022 interaction with the SARS-CoV spike protein by negative-stain EM and cryo-EM. Three CR3022 Fabs bind per trimer with the RBD observed in different up-conformations due to considerable flexibility of the RBD. In one of these conformations, quaternary interactions are made by CR3022 to the N-terminal domain (NTD) of an adjacent subunit. Overall, this study provides insights into antigenic variation and potential cross-neutralizing epitopes on SARS-like viruses.
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SciScore for 10.1101/2020.09.21.305441: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Briefly, MLV-gag/pol and MLV-CMV plasmids was co-transfected into HEK293T cells along with full-length or P384A SARS-CoV-2 spike plasmids using Lipofectamine 2000 to produce pseudoviruses competent for single-round infection. HEK293Tsuggested: NoneAt 42 to 48 hours post-infection, HeLa-hACE2 cells were lysed using 1x luciferase lysis buffer (25 mM Gly-Gly pH 7.8, 15 mM MgSO4, 4 mM EGTA, and 1% Triton X-100). HeLa-hACE2suggested: NoneSoftware and Algorithms Sentences Resources Structures were solved by molecular replacement using PHASER [43] with PDB 6W41 for CR3022 Fab [20] and PDB … SciScore for 10.1101/2020.09.21.305441: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Briefly, MLV-gag/pol and MLV-CMV plasmids was co-transfected into HEK293T cells along with full-length or P384A SARS-CoV-2 spike plasmids using Lipofectamine 2000 to produce pseudoviruses competent for single-round infection. HEK293Tsuggested: NoneAt 42 to 48 hours post-infection, HeLa-hACE2 cells were lysed using 1x luciferase lysis buffer (25 mM Gly-Gly pH 7.8, 15 mM MgSO4, 4 mM EGTA, and 1% Triton X-100). HeLa-hACE2suggested: NoneSoftware and Algorithms Sentences Resources Structures were solved by molecular replacement using PHASER [43] with PDB 6W41 for CR3022 Fab [20] and PDB 2AJF for SARS-CoV RBD [44]. PHASERsuggested: (Phaser, RRID:SCR_014219)Iterative model building and refinement were carried out in COOT [45] and PHENIX [46], respectively. COOTsuggested: (Coot, RRID:SCR_014222)PHENIXsuggested: (Phenix, RRID:SCR_014224)Ramachandran statistics were calculated using MolProbity [47]. MolProbitysuggested: (MolProbity, RRID:SCR_014226)Micrographs were collected using Leginon [48] and images were transferred to Appion [49] for particle picking using a difference-of-Gaussians picker (DoG-picker) [50] and generation of particle stacks. Leginonsuggested: (Leginon, RRID:SCR_016731)Select 3D classes were auto-refined on Relion and used for making figures using UCSF Chimera [52]. Relionsuggested: (RELION, RRID:SCR_016274)Micrographs were collected through Leginon software at a nominal defocus range of -0.4 µm to -1.6 µm and MotionCor2 was used for alignment and dose weighting of the frames [48, 53]. MotionCor2suggested: (MotionCor2, RRID:SCR_016499)Micrographs were transferred to CryoSPARC 2.9 for further processing [54]. CryoSPARCsuggested: (cryoSPARC, RRID:SCR_016501)CTF estimations were performed using GCTF and micrographs were selected using the Curate Exposures tool in CryoSPARC based on their CTF resolution estimates (cutoff 5 Å) for downstream particle picking, extraction and iterative rounds of 2D classification and selection [55]. GCTFsuggested: (GCTF, RRID:SCR_016500)Calculation of rotation angles: Comparisons of subunit rotation angles among different structures were performed with a software “Superpose” in the CCP4 package [56, 57]. CCP4suggested: (CCP4, RRID:SCR_007255)Results from OddPub: Thank you for sharing your data.
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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